The SMART assay. (a) Specific probes hybridise with the target to form a three-way junction (3WJ), assisted by facilitator probes (f1 & f2). The 3WJ initially contains a single-stranded, inactive T7 RNA polymerase promoter sequence. The promoter is made double stranded (active) by extension (by Bst DNA polymerase) off the 3' of the extension probe, leading to the generation of large amounts of RNA signal (by T7 RNA polymerase), which may itself be amplified if required. (b) Detection of RNA signal by ELOSA (Enzyme Linked OligoSorbant Assay). The assay uses 2 specific probes: a biotinylated capture probe and enzyme (Alkaline phosphatase, AP) linked detection probe. Non-specific nucleic acid and 3WJ probes are removed, following binding in a streptavidin coated well, and RNA signal is detected via a colour change. Quantification of signal takes place in a 96 well plate, allowing multiple samples to be analysed simultaneously.